Methods of incerasing protein expession levels

ABSTRACT

Abstract of the Disclosure 
     The present invention relates to methods of increasing protein expression levels whereby at least one amino acid in a protein amino acid sequence is substituted for the amino acid, proline. Preferably, the substitution occurs within 15 amino acids, more preferably within 10 amino acids and most preferably within 5 amino acids of a cysteine amino acid residue. The present invention not only includes methods for polypeptides with proline substitutions, but also polynucleotides with codon substitutions for which a codon for any amino acid, except proline, is substituted for a codon encoding for proline.

Detailed Description of the Invention Summary of the Invention

The present invention relates to methods of increasing proteinexpression levels whereby at least one amino acid in a protein aminoacid sequence is substituted for the amino acid, proline. Preferably,the substitution occurs within 15 amino acids, more preferably within 10amino acids and most preferably within 5 amino acids of a cysteine aminoacid residue. The present invention not only includes methods forpolypeptides with proline substitutions, but also polynucleotides withcodon substitutions for which a codon for any amino acid, exceptproline, is substituted for a codon encoding for proline. Specifically,the codons encoding proline are CCU, CCC, CCA and CCG for RNA and CCT,CCC, CCA, and CCG for DNA.

Any protein of interest can be included in the invention, provided thatit can be expressed from a recombinant DNA molecule in a suitable hostcell and remain functionally active. Preferably, the protein is onewhose expression is limited by constraints on conformational folding.More preferably, the protein will contain one or more cysteine aminoacid residues and may require the formation of correct disulfide bonds,resulting in proper conformational folding of the tertiary structure ofthe polypeptide.

The present invention solves, in part, the continuing need in the artfor means to improve the efficiency of expression of recombinantproteins in mammalian and other host cells. The invention providesmethods that overcome the problem of inefficient folding ofpolypepetides, which limits the expression of certain heterologouspolypeptides , and provides for higher levels of expression of suchpolypeptides.

Brief Description of the Figures

Figure 1A represents a diagramatic representation of a flow cytometricanalysis of yeast cells stained with a polyclonal antibody directedagainst TNFrED and R-phycoerythrin-conjugated donkey anti-goat IgG.Yeast cells contained either the pYES2 plasmid (left panel) or thepYES2-TNFrED-Agg plasmid (right panel).

Figure 1B is a diagramatic representation of a flow cytometric analysisof yeast cells stained with biotinylated hTNF-( and FITC-labeled avidin.Yeast contained either the pYES2 plasmid (left panel) or thepYES2-TNFrED-Agg plasmid (right panel).

Figure 1C is a diagramatic representation of a two-color flow cytometricanalysis of yeast cells that have been stained with biotinylated hTNF-(and FITC-labeled avidin and a polyclonal antibody directed againstTNFrED and R-phycoerthrin-conjugated donkey anti-goat IgG. Yeast weretransformed with a mutant library (left panel) or with the same libraryafter three rounds of sorting (right panel).

Figure 1D is a diagramatic representation of a flow cytometric analysisof yeast cells stained with biotinylated hTNF-( and FITC-labeled avidin.In the left panel the shaded histogram represents the yeast expressingwild-type TNFrED whereas the unshaded histogram represents clone 11. Inthe right panel the shaded histogram represents the yeast expressingwild-type TNFrED whereas the unshaded histogram represents clone 6.

Figure 2 is a diagramatic representation of a saturation bindinganalysis of yeast expressing wild-type TNFrED (Fig 2A), mutant clone 6(Fig 2B)or 11 (Fig 2C). Triplicate samples of yeast were incubated withincreasing concentrations of [¹²⁵I]TNF-(. Binding experiments wererepeated two-three times with similar results.

Figure 3 is a diagramatic representation of the (-( plot of thesubstituted residues in the mutant clones 6 and 11 and the correspondingresidues found in wild-type TNFrED wherein the shaded regions are thefavorable conformations obtained from an analysis of 136 non-homologousprotein structures at a resolution of 1.8 Å or higher. The darker thecolor, the higher the frequency of the (-( angles of the residues, andhence the more favored conformation. The (-( angles of the correspondingresidue in the TNFrED crystal structures are shown in red squares(1ncf.pdb, TNFrED dimer structure, 1.85 Å resolution), red diamonds(1ext.pdb, TNFrED dimer structure, 2.85 Å resolution), and red triangles(1tnr.pdb, TNF/TNFrED complex structure, 2.85 Å resolution). Therepresentative high-resolution dataset was used to generate the greensymbols and these symbols represent the (-( angles of the substitutedresidue in the same tri- (green ‘x’s) or tetra- (green circles) aminoacid context as that found in wild-type TNFrED or the mutant clones (seealso Table 4). (A). Ser87. (B) Pro87. (C). His34. (D) Pro34. (E). Ser57.(F). Ile57.

Figure 4 presents the amino acid and polynucleotide sequences of TNFrED.

Detailed Description of the Invention

The present invention is based on the unexpected finding that thesubstitution of one or more amino acids for the amino acid proline inthe amino acid sequence of a polypeptide results in higher levels ofexpression of that protein. While not wishing to be bound by theory,evidence supports the suggestion that the biochemical mechanism allowinghigher levels of expression relates to the promotion of properconformational folding of a protein.

Through screening of mutant clones of the extracellular domain of thereceptor for Tissue Necrosis Factor (TNFrED) with the yeast displaysystem we were able to identify mutations of TNFrED that conferred ahigher expression level in yeast when compared to wild-type TNFrED. Oneclone contained the S87P mutation and the other clone contained the H34Pand S57I mutations. These mutations did not change the affinity ofTNFrED for TNF-(. Expression of these mutants in mammalian cellsgenerated similar findings, strongly suggesting that the mechanismthrough which the mutations increase expression is conserved betweenyeast and mammalian cells. Examination of the mutations individually orin combination revealed that either proline change increased theexpression of TNFrED whereas the S57I mutation had no effect onexpression.

In each mutant clone, we showed that the residue responsible for thehigher protein expression levels is a proline substitution next to acysteine involved in a disulfide bond. In proline the nitrogen atom ispart of a rigid ring and no rotation of the ring N-C bond is possible.Thus the choices of (-( angles are fewer in the proline substitutionsthan the histidine or serine residues found in the wild-type TNFrED(shaded areas in Fig. 3). Moreover, the (-( angles of the histidine orserine residue found in the crystal structures of TNFrED are located inthe more favored regions of the (-( plot of a proline residue. Theseresults indicate that proline is the preferred residue at position 34 or87 in terms of the main-chain conformation. This phenomenon is moreevident in the uncomplexed structure of TNFrED with the highestresolution (pdb code: 1ext; see the red filled squares in Figure 3).Therefore, the mutations have no advantage in the complexed formcompared to the uncomplexed form, which is in agreement with ourobservation that the mutants do not affect the affinity of TNFrED forligand. To test if there is any change of the favorable status of aproline for the given (-( angles when the adjacent one or two aminoacids are also considered, we searched the representative PDB datasetfor the same sequences in the mutated form and the wild-type form. Thereare more cases of mutated sequences than the wild-type sequences (seeTable 3 and also the green ‘x’s in Figure 3). These results furtheremphasize that proline is the preferred residue at position 34 or 87,both in terms of the main-chain conformation for that particular residueand also for when the surrounding residues and structure environment aretaken into consideration. The S57I mutation is a different situation. Itresults in a narrower region of (-( angles but deviates away from thepreferred (-( angle region to a slightly less preferred region (Fig. 3).Thus, there appears to be no advantage of the S57I mutation in term of(-( preference at that position. This is consistent with ourexperimental observation that the presence of the S57I mutation alonedoes not significantly alter the expression level of TNFrED. Taking allthe data together, we conclude that introduction of the proline residuesassists the local sequence of each mutant to adopt the conformationsseen in crystal structures of TNFrED thereby fixing neighboring cysteineresidues into the correct orientation. We proposed that properorientation of the cysteine in turn facilitates formation of the correctdisulfide bond and results in a higher yield of correctly foldedmolecules. Our finding that these proline substitutions increase theexpression level of TNFrED is consistent with this proposal.Furthermore, proline substitutions can be extended to other proteinswhere the formation of disulfide bonds is thought to be a limitingfactor in expression.

The present invention relates to methods of increasing proteinexpression levels whereby at least one amino acid in a protein aminoacid sequence is substituted for the amino acid, proline. Preferably,the substitution in these proline-substituted polypeptides occurs within15 amino acids, more preferably within 10 amino acids and mostpreferably within 5 amino acids of a cysteine amino acid residue. Thepresent invention not only includes methods for polypeptides withproline substitutions, but also polynucleotides with codon substitutionsfor which a codon for any amino acid, except proline, is substituted fora codon encoding for proline. Specifically, the codons encoding prolineare CCU, CCC, CCA, CCG for RNA and CCT, CCC, CCA, CCG for DNA (see Table1). I. Definitions:

In general, the following words or phrases have the indicated definitionwhen used in the description, examples and claims.

“Protein” shall mean any polypeptide comprised of amino acids and havinga unique amino acid sequence. The term “protein” may be usedinterchangeably herein with the term “polypeptide”.

“Substitution” shall mean the introduction of an amino acid, either byreplacement of an existing amino acid residue or by insertion of anadditional residue. In the present invention, the replacement orinserted amino acid is proline.

“Increased Expression” shall mean higher or greater expression levels ofprotein when comparing the expression levels of a protein with itsoriginal amino acid sequence with the expression levels of the sameprotein with one or more proline substitutions.

“Amino Acid” shall mean an amino acid residue contained in the groupconsisting of the 20 naturally occurring amino acids. In the presentapplication, amino acid names are used as defined by the ProteinDataBank (PDB) (www.pdb.org), which is based on the IUPAC nomenclature(IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residuenames), Eur. J. Biochem., 138, 9-37 (1984) together with theircorrections in Eur. J. Biochem., 152, 1 (1985); i.e. alanine (Ala or A),cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (GLU or E),phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H),isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine(Met or M), asparagine (Asn or S), proline (Pro or P), glutamine (Gln orQ), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine(Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

“Positioned Within” shall mean being positioned proximal to a particularreference amino acid in an amino acid sequence, in either the 3’ or 5’direction.

“Codon” shall mean a triplet of nucleotides coding for a single aminoacid. (see Table 1)

“Polynucleotide” shall mean a single stranded molecule of DNA or RNA.

The terminology used for identifying amino acid positions /substitutions is illustrated as follows: H34P indicates that positionnumber 34 in the linear sequence of amino acids for a particular proteinas shown in Figure 4 and SEQ ID NO: 1 is occupied by a histidine residueand that it has been substituted with a proline residue. Unlessotherwise indicated, the numbering of amino acid residues made herein ismade relative to the amino acid sequence shown in Figure 4 and SEQ IDNO: 1 (for Tissue Necrosis Factor). Multiple substitutions are indicatedwith a “+”, e.g., H34P + S57I means an amino acid sequence whichcomprises substitution of the histidine residue in position 34 by aproline residue and substitution of the serine residue in position 57 bya isoleucine residue. Table 1. The Genetic Code (RNA to Amino Acids)*First Third Position Position (5’ end) Second Position (3’ end) U C A GU PhePheLeuLeu SerSerSerSer TyrTyrStop (och) CysCysStopTrp U C A G Stop(amb) C LeuLeuLeuLeu ProProProPro HisHisGlnGln ArgArgArgArg U C A G(Met) A IleIleIleMet ThrThrThrThr AsnAsnLysLys SerSerArgArg U C A G(start) G ValValValVal AlaAlaAlaAla AspAspGluGlu GlyGlyGlyGly U C A G(Met

Methods for Generating an Expression System for Proline SubstitutedPolypeptides:

The proline substituted polypeptides of the present invention may beproduced by any suitable method known in the art. These methods includethe construction of nucleotide sequences encoding the respective prolinesubstituted polypeptides and expressing the amino acid sequence in asuitable transfected host. Proline substituted polypeptides of thepresent invention may also be produced by chemical synthesis or by acombination of chemical synthesis and recombinant DNA technology.

Proline substituted polypeptides of the invention may be constructed byisolating or synthesizing a nucleotide sequence encoding the parent. Thenucleotide sequence is then changed so as to effect the substitution ofthe relevant amino acid residue(s). The nucleotide sequence can bemodified by site directed mutagenesis as in the Examples of the presentspecification. In the alternative, the nucleotide sequence may beprepared by chemical synthesis, wherein oligonucleotides are designedbased on the specific amino acid sequence of the proline susbstitutedpolypeptides.

The nucleotide sequence encoding the proline susbstituted polypeptide isinserted into a recombinant vector and operably linked to controlsequences necessary for expression of the polypeptide in the desiredtransfected host cell. One of skill in the art may make a selectionamong these vectors, expression control sequences and hosts withoutundue experimentation.

The recombinant vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector is one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the nucleotidesequence encoding the polypeptide of the invention is operably linked toadditional segments required for transcription of the nucleotidesequence. The vector is typically derived from plasmid or viral DNA. Anumber of suitable expression vectors for expression in the host cellsmentioned herein are commercially available or described in theliterature

The recombinant vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. An example of such asequence, (when the host cell is a mammalian cell) is the SV40 origin ofreplication.

The vector may also comprise a selectable marker, e.g. a gene whoseproduct complements a defect in the host cell, such as the gene codingfor dihydrofolate reductase (DHFR) or one which confers resistance to adrug, e.g. ampicillin, kanamycin, tetracycline chloramphenicol,neomycin, hygromycin or methotrexate.

The vector may also comprise an amplifiable gene, such as DHFR, suchthat cells having multiple copies of the amplifiable gene and flankingsequences, including the proline substituted polypeptide DNA, can beselected for on appropriate media.

The term "control sequences" is defined herein to include all componentswhich are necessary or advantageous for the expression of thepolypeptide of the invention. Examples of suitable control sequences fordirecting transcription in mammalian cells include the early and latepromoters of SV40 and adenovirus, e.g. the adenovirus 2 major latepromoter, the MT-1 (metallothionein gene) promoter and the humancytomegalovirus immediate-early gene promoter (CMV).

The nucleotide sequences of the invention encoding the prolinesubstituted polypeptides, whether prepared by site-directed mutagenesis,synthesis, PCR or other methods, may optionally also include anucleotide sequence that encodes a signal peptide. The signal peptide ispresent when the polypeptide is to be secreted from the cells in whichit is expressed. Such signal peptide, if present, should be onerecognized by the cell chosen for expression of the polypeptide. Thesignal peptide may be homologous (e.g. be that normally associated withthe parent polypeptide) or heterologous (i.e. originating from anothersource than parent polypeptide) to the polypeptide or may be homologousor heterologous to the host cell, i.e. be a signal peptide normallyexpressed from the host cell or one which is not normally expressed fromthe host cell.

Any suitable host may be used to produce the proline susbstitutedpolypeptide of the invention, including bacteria, fungi (includingyeasts), plant, insect, mammal, or other appropriate animal cells orcell lines, as well as transgenic animals or plants. Examples ofsuitable mammalian host cells include Chinese hamster ovary (CHO) celllines, (e.g. CHO-KL; ATCC CCL-61), Green Monkey cell lines (COS) (e.g.COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NSIO),Baby Hamster Kidney (BI-EK) cell lines (e.g. ATCC CRL-1632 or ATCCCCL-10), and human cells (e.g. BEK 293 (ATCC CRL-1573)), as well asplant cells in tissue culture. Additional suitable cell lines are knownin the art and available from public depositories such as the AmericanType Culture Collection, USA. Methods for introducing exogeneous DNAinto mammalian host cells include calcium phosphate-mediatedtransfection, electroporation, DEAE-dextran mediated transfection,liposome-mediated transfection and viral vectors.

Cells are cultivated in a nutrient medium suitable for production of theproline susbstituted polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation,small-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermenters performed in a suitable medium and under conditions allowingthe proline susbstituted polypeptide to be expressed and/or isolated.The cultivation takes place in a suitable nutrient medium comprisingcarbon and nitrogen sources and inorganic salts, using procedures knownin the art. Suitable media are available from commercial suppliers ormay be prepared according to published compositions (e.g. in cataloguesof the American Type Culture Collection). If the proline susbstitutedpolypeptide is secreted into the nutrient medium, it can be recovereddirectly from the medium. If the proline susbstituted polypeptide is notsecreted, it can be recovered from cell lysates.

The resulting proline substituted polypeptide may be recovered bymethods known in the art. For example, it may be recovered from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray drying, evaporation,or precipitation.

The proline substituted polypeptides may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g. ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.preparative isoelectric focusing), differential solubility (e.g.ammonium sulfate precipitation), SDS-PAGE, or extraction.

Pharmaceutical composition of the invention and its use

In one aspect, the proline susbstituted polypeptide or thepharmaceutical composition according to the invention is used for themanufacture of a medicament for treatment of diseases, disorders orconditions.

In another aspect the proline susbstituted polypeptide or thepharmaceutical composition according to the invention is used in amethod of treating a mammal, in particular a human, comprisingadministering to the mammal in need thereof such proline susbstitutedpolypeptide or pharmaceutical composition thereof.

It will be apparent to those of skill in the art that an effectiveamount of a conjugate, preparation or composition of the inventiondepends, inter alia, upon the disease, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in conjunction with other therapeutic agents, theserum half-life of the compositions, and the general health of thepatient. Typically, an effective dose of the preparation or compositionof the invention is sufficient to ensure a therapeutic effect.

The proline susbstituted polypeptide produced by the methods of theinvention is normally administered in a composition including one ormore pharmaceutically acceptable carriers or excipients."Pharmaceutically acceptable" means a carrier or excipient that does notcause any untoward effects in patients to whom it is administered. Suchpharmaceutically acceptable carriers and excipients are well known inthe art, and the polypeptide or conjugate of the invention can beformulated into pharmaceutical compositions by well-known methods (seee.g. Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro,Ed., Mack Publishing Company (1990); Pharmaceutical FormulationDevelopment of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds.,Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients, 3rdedition, A. Kibbe, Ed., Pharmaceutical Press (2000)). Pharmaceuticallyacceptable excipients that may be used in compositions comprising thepolypeptide or conjugate of the invention include, for example,buffering agents, stabilizing agents, preservatives, isotonifiers,nonionic surfactants or detergents (“wetting agents"), antioxidants,bulking agents or fillers, chelating agents and cosolvents.

The pharmaceutical composition of the proline susbstituted polypeptideof the invention may be formulated in a variety of forms, includingliquids, e.g. ready-to-use solutions or suspensions, gels, lyophilized,or any other suitable form, e.g. powder or crystals suitable forpreparing a solution. The preferred form will depend upon the particularindication being treated and will be apparent to one of skill in theart.

The pharmaceutical composition containing the proline susbstitutedpolypeptide of the invention may be administered intravenously,intramuscularly, intraperitoneally, intradermally, subcutaneously,sublingualy, buccally, intranasally, transdermally, by inhalation, or inany other acceptable manner, e.g. using PowderJect@ or ProLease(Dtechnology or a pen injection system. The preferred mode ofadministration will depend upon the particular indication being treatedand will be apparent to one of skill in the art. In particular, it isadvantageous that the composition be administered subcutaneously, sincethis allows the patient to conduct the self-administration.

The pharmaceutical composition of the invention may be administered incon-junction with other therapeutic agents. These agents may beincorporated as part of the same pharmaceutical composition or may beadministered separately from the polypeptide or conjugate of theinvention, either concurrently or in accordance with any otheracceptable treatment schedule. In addition, the polypeptide, conjugateor pharmaceutical composition of the invention may be used as an adjunctto other therapies.

Exemplification: Enhanced Expression of Tissue Necrosis Factor (TNF)

The extracellular domain of the p55 TNF receptor (TNFrED) was randomlymutated and libraries of TNFrED mutants were displayed on the surface ofyeast cells. Two mutant TNFrED clones were identified byfluorescence-activated cell sorting (FACS) that expressed two- tofive-fold higher in yeast compared to wild-type TNFrED. In one mutantclone there was a Ser to Pro change at position 87 and in the othermutant clone there was a His to Pro change at position 34 and a Ser toIle change at position 57. The presence of either the S87P or H34Pmutation resulted in higher expression levels of TNFrED in HEK293-EBNAcells whereas the S57I mutation had no effect on expression. Thesesubstituted residues did not have an effect on the affinity for TNF-(.Examination and analysis of the substituted residues in the crystalstructures of TNFrED indicates that the introduction of proline residueslikely assists the local sequence of the mutants to adopt favorableconformations that fix the neighboring cysteine residues into thecorrect orientation for proper disulfide bond formation. Thisfacilitation of the formation of selected disulfide bonds then resultsin a higher yield of correctly folded molecules in both yeast andmammalian cells.

Example 1: Expression of functional TNFrED on the surface of S.cerevisae

The extracellular domain of the p55 TNF receptor (TNFrED) was fused tothe C-terminal portion of (-agglutinin and expressed in the S cerevisaestrain BJ2168 (a, prc1-407, prb1-1122, pep4-3, leu2, trp1, ura3-52)using the pYES2 vector. The C-terminal portion of (-agglutinin istightly anchored in the cell wall and serves as a scaffold to presentTNFrED on the cell surface. To facilitate the transport of theTNFrED-agglutinin fusion protein into the secretory pathway, the signalsequence of TNF receptor was replaced with the yeast invertase signalsequence. The TNFrED-agglutinin fusion gene was under the regulation ofthe inducible GAL1 promoter. Switching the carbon source of the yeastculture from glucose to galactose resulted in induction of the GAL1promoter and expression of TNRrED-agglutinin. In a flow cytometricanalysis with polyclonal antibodies directed against TNFrED we foundthat in an induced culture approximately 70% of the yeast expressedTNFrED-agglutinin on the cell surface (Fig. 1A). To determine whetherTNFrED on the yeast cell surface was folded correctly and could bindTNF-( we performed a flow cytometric analysis using biotinylated TNF-(as a probe and FITC-labeled avidin as the detection reagent. Yeastexpressing the TNFrED-agglutinin fusion gene bound more biotinylatedTNF-( than did yeast containing the pYES2 vector (Fig. 1B). Addingexcess unlabeled TNF-( (data not shown) reversed the shift in thehistogram seen with the yeast expressing the TNFrED-agglutinin fusiongene.

Example 2: Selection of mutant TNFrED clones with enhancedbiotinylated-TNF-( binding.

A modification of the mutagenesis approach previously described(Hermes,J.D., Parekh,S.M., Blacklow,S.C., Koster,H. & Knowles,J.R. Areliable method for random mutagenesis: the generation of mutantlibraries using spiked oligodeoxyribonucleotide primers. Gene 84,143-151 (1989)) was used to generate random mutant libraries. In thisapproach mutant oligonucleotides are produced by spiking a predeterminedlevel of the “wrong” nucleotides at each position. The level ofcontamination of the wrong nucleotides was adjusted to generate eitheran average of two or three random point mutations per oligonucleotide.Each mutant library covered between 40 to 105 base pairs of TNFrED. Tenrandom clones from each library were sequenced. The type and theposition of mutations were random and the regions were found to containthe anticipated average number of mutations. Approximately 40-50% of theclones contained either a small deletion or insertion, which results inthe expression of a truncated TNFrED. Presumably these deletions orinsertions resulted from errors incorporated during the oligonucleotidesynthesis process. The size of each library was between 0.5 x 10⁶ and 10x 10⁶ independent mutant clones. Each library was transformed into thestrain BJ2168 and approximately 1 x 10⁶ independent transformants wereselected for binding to both biotinylated-TNF-( and polyclonalantibodies directed against the TNFrED. The window of thetwo-dimensional fluorescence histogram that was chosen to select for thesubpopulation of yeast expressing active TNFrED is shown in Fig. 1C. Theselected subpopulation of yeast was grown and reselected with two-colorsorting. After several rounds of two-color sorting the population ofyeast in the selected window was enriched (Fig. 1C). Following three tofour rounds of cell sorting, individual clones were analyzed byexamining binding of biotinylated-TNF-( to the TNFrED on the cellsurface. A majority of the clones analyzed from each sorted libraryappeared to bind higher levels of biotinylated-TNF-(. The mutant TNFrEDplasmid was recovered from each yeast clone and re-transformed into theBJ2168 strain and then analyzed by flow cytometry for binding ofbiotinylated-TNF(. The vast majority of yeast clones were falsepositives as the recovered plasmids did not confer higher levels ofbiotinylated-TNF-( binding. When false positive yeast clones wereanalyzed in the absence of biotinylated TNF-( and avidin-FITC, they werefound to be shifted as compared to the parental BJ2168 strain. Thesefalse positive yeast clones are approximately 30% larger in sizecompared to the parental BJ2168, which is presumably what gives rise tothe shift in the baseline absorbance. However, two mutant clones, 6 and11, from different libraries were identified that when transformed intothe BJ2138 strain conferred higher levels of binding of biotinylated TNFand therefore are true positives (Fig. 1D).

Example 3: Characterization of mutant clones 6 and 11.

The TNFrED coding region in mutant clones 6 and 11 was sequenced and, asanticipated, the mutations for each clone were only found in thesequence region mutated for that specific library. In mutant clone 11there was a point mutation that results in Ser to Pro change at position87 and in mutant clone 6 there were two point mutations that result inan His to Pro change at position 34 and a Ser to Ile change at position57.

To determine whether the mutations increase binding of biotinylated TNFthrough increased expression of TNFrED or by increasing the affinity ofTNFrED for TNF, we performed a saturation binding experiment on yeastexpressing either mutant clone 6 or 11. Analysis of saturation bindingexperiments (Fig. 2) indicated that yeast expressing either mutant clone6 or 11 express higher levels of functional TNFrED than wild-type TNFrEDand that the presence of these mutations does not affect the affinity ofTNFrED for TNF-(. The approximate number of receptors/cell for yeastexpressing mutant clone 6, mutant clone 11 and wild-type TNFrED was3930, 1490, 740, respectively.

Example 4: Expression of mutated TNFrEDs in mammalian cells.

Next, it was determined whether characteristics of the mutant clonesderived in yeast are similar in mammalian cells. Toward this goal weconstructed a mammalian expression vector containing the human growthhormone signal sequence fused at the amino terminus to the sequenceencoding mature TNFrED. We have previously found that the human growthhormone signal sequence is more efficient in inducing the secretion ofTNFrED than the native signal sequence. Site-directed mutagenesis wasused to incorporate each mutation found in mutant clones 6 and 11,either individually or in various combinations. The TNFrED mutants weretransiently expressed in HEK293-EBNA cells and the amount of secretedTNRrED was quantitated with an ELISA specific for TNFrED. The results(Table 2) indicated that either mutation H34P or S87P increased theexpression level of TNFrED. Moreover, the relative increase inexpression is similar to what is seen when these mutants are expressedin yeast (Fig. 2). The S57I mutation alone did not alter the expressionlevel of TNFrED (Table 2). The effects of these mutations do not appearto be additive as the presence of both H34P and S87P on the sameconstruct did not increase the level of TNFrED secreted in comparison tothat found with H34P alone.

The kinetics of TNF binding to the various mutants of TNFrED by surfaceplasmon resonance in a BIAcore instrument was also measured. The resultsin Table 2 indicate no difference between wild type and mutant TNFrEDstested with respect to TNF-( binding. As indicated in ExperimentalProtocol, we use mild regeneration conditions and typically collect30-40 cycles of data on every surface. We averaged results obtained onthree different surfaces to obtain the means (and 95% confidenceintervals) of parameters for the control. The principal error introducedby this manner of data acquisition is an increase in the variance offitted parameters for controls when data are pooled across differentsurfaces and different runs (see result for purified TNFrED in Table 3).Nevertheless, the data are good enough to detect a 15% change in the kon, a 33% change in koff , or both. This is evidenced in the results weshow for negative control and HBS (Hepes buffered saline) data collectedafter 27 and 36 cycles were acquired on the respective surfaces. Thiswas done to illustrate the worst case scenario since no TNF-( bindingcould be measured when these "ligands" (negative control, conditionedmedium, and HBS) were tested at the beginning of a run. The residue froma 36 cycle buildup of various partially denatured TNF receptor proteinsdemonstrates strikingly different kinetics from intact control, wildtype or any mutated TNF receptor. Consistent with our findings from theyeast expression studies, the presence of H34P, S87P or S57I mutationsdoes not alter the affinity of TNFrED for TNF-(. Table 2. TransientTransfection of HEK293-EBNA cells with wild-type TNFrED and mutants ofTNFrED. DNA ng/ml of TNFrED Fold over wild-type wild-type 239 + 5.0 –H34P 724 + 40 3 S57I 190 + 19 0.8 S87P 361 + 21 1.5 H34P, S57I 614 + 542.6 H34P, S87P 711 + 33 3 H34P, S57I, S87P 704 + 26 2.9

HEK293-EBNA cells were transiently transfected with plasmid DNA. 48 hpost-transfection the conditioned medium was collected and the amount ofTNFrED was quantitated with an ELISA. The amount of TNFrED is the meanof four dishes + the standard deviation. This experiment was repeatedthree times and similar results were obtained. A single representativeexperiment is shown. Table 3. Binding Kinetics and TNF Affinity ofTNF-rED mutants. Sample k_(on × 10) ^(6 M) ^(1 s) ⁻¹ k_(off × 10)^(−3 s) ⁻¹ K_(D × 10) ^(−9 M) R_(max RU) (² wild-type TNFrED 2.0 0.8 0.488.9 7.2 H34P 1.9 0.9 0.5 88.5 7.6 S571 1.9 0.9 0.5 85.4 7.0 S87P 2.01.1 0.6 90.3 4.7 H34P + S571 1.9 1.2 0.6 84.2 3.9 H34P + S87P 1.9 1.20.6 86 4.9 H34P + S87P + S571 1.8 1.0 0.6 82 4.7 negative control 1.32.0 1.6 25.1 0.9 HBS 1.3 1.9 1.5 24.2 0.8 purified TNFrEDAvg 2.0 (0.30.9 (0.3 0.5 (0.1 77.5 (3 (95% C.I.

Each TNFrED and control sample listed was analyzed with seven differentconcentrations of TNF-( (see Experimental Protocol). For a seven leveldata set k_(on), k_(off) and R_(max) were fit globally using a modelwhich incorporates baseline drift. The negative control (conditionedmedium) and the HBS control show K_(d) values of ~1.5nM due to the mildregeneration conditions used (see Experimental Protocol). Under theseconditions, we routinely note analyte-independent 0.5-1.0 RU/cycleupward movement in the absolute value of Rpoint baseline (data notshown). We suspected this result was due to small amounts of partiallydenatured TNFrED and mutant TNFrED remaining after each regenerationtreatment and checked to see that it did not depend upon the nature ofthe mutant (data not shown). Analysed prior to a 27-36 cycle run of datacollection, negative controls showed no TNF-( binding.

Example 5: Examination of the locations of substituted residues incrystal structures of TNFrED

The proline substitutions in mutant clone 6 and 11 are adjacent tocysteine residues (cysteines 33 and 88) involved in disulfide bonds. Toanalyze whether there is a potential structural role for the mutantresidues, we tabulated the conformational preferences of proline,serine, histidine and isoleucine residues in the crystal structures ofTNFrED (pdb codes: 1ext, 1ncf and 1tnr) and in general proteinstructures. Since the conformation of polypeptides is controlled by the(-( angles of the residues, we plotted the (-( distribution of thoseresidues in the dataset of high-resolution and non-homologous proteinstructures. As shown in Figure 3, the (-( distribution of a prolineresidue is more restricted than that of the other three residues, andthe (-( distribution of a serine residue is greater than that of anisoleucine residue. Specifically, the size ratio of the distributionsfor serine to proline is 2.5, for histidine to proline is 1.8, and forserine to isoleucine is 1.4. Moreover, the (-( angles of S87 and of H34in TNFrED crystal structures (red filled marks) preferentially overlapthe favored region of proline’s (-( plot as compared to the favoredregion of (-( plots for serine or histidine. This preference is slightlymore noticeable in the crystal structures of TNFrED itself (pdb code:1ext; resolution 1.85 A [red squares], or 1ncf; resolution 2.85 A [reddiamonds]) than in the crystal structure of the TNF/TNFrED complex (pdbcode: 1tnr; resolution 2.85 A [red triangles]). In contrast, the (-(angles of Ser57 in TNFrED crystal structures are located in a slightlymore favored region in the serine (-( plot compared to that found withisoleucine, although the strongly favored region of either residue doesnot significantly overlap the distribution taken from crystalstructures.

To assess the effect of adjacent residues in the choice of (-(preferences by the substituted residue, we analyzed the (-( angles inthe pdb database representative set containing the same tri- (green‘x’s) or tetra- (green circles) peptide fragments as those in clone 6,clone 11 or wild-type TNFrED. As shown in Figure 3 and listed in Table4, there are more cases of proline residues in which the (-( angles aresimilar to those of H34 or S87 in the TNFrED crystal structures thanthere are of histidine or serine residues with (-( angles similar tothose of H34 or S87. The data are consistent with the (-( angles of S57in the TNFrED crystal structures being slightly more favored for aserine residue than for an isoleucine. Table 4. List of crystalstructures containing the same tri- or tetra- amino acid sequence asthose in the wild-type TNFrED or mutant clones. (a). The criteria forsimilar (-( angles is that the (-( angle of the subject residue iswithin 15 degree of the (-( angles found in the corresponding residue ofany of the TNFrED crystal structures. (b). The residues in bold areeither the substituted residue in the mutant clone or the residue foundin wild-type TNFrED.

Experimental Protocols Used in the Examples

Plasmids.

The TNFrED-agglutinin fusion was constructed by linking the signalsequence from the invertase gene to the hTNFrED sequence encodingresidues 12 to 172 which was then fused to the C-terminal portion of the(-agglutinin gene encoding residues 330 to 650 (1, 27). Between TNFrEDand the (-agglutinin gene was a sequence encoding the flexible linkerGQPAAAPA. This linker is similar to the sequence of hinges betweendomains in immunoglobulins . The TNFrED-agglutinin fusion gene wassubcloned into the pYES2 vector (Invitrogen Corp.) to generatepYES2-TNFrED-Agg for expression in yeast.

The hTNFrED coding sequence from residue 12 to residue 172 was subclonedinto pCMV, a mammalian expression vector, to generate pCMV-TNFrED. InpCMV-TNRrED the human growth hormone signal sequence is fused upstreamof the hTNFrED sequence. The expression of hTNFrED in pCMV is regulatedby the human cytomegalovirus (CMV) immediate-early enhancer/promoterregion. Downstream of hTNFrED is the human growth hormonepolyadenylation signal. pCMV-TNFrED containing either the mutation S57Ior S87P was generated by subcloning the appropriate region from mutantclones 6 and 11, respectively. pCMV-TNFrED containing the H34P mutationwas generated using the whole vector PCR technique (6). Combinations ofthe mutations of TNFrED (H34P+S57I, H34P+S87P, H34P+S57I+S87P) wasaccomplished by subcloning the appropriate regions. The sequence of thecoding region of all constructs was verified with the Thermo Sequenaseradiolabeled terminator cycle sequencing kit (Amersham PharmaciaBiotech). Expression of TNFrED on the surface of yeast cells.

Saccharomyces cerevisiae strain BJ2168 (a, prc1-407, prb1-1122, pep4-3,leu2, trp1, ura3-52; Yeast Genetic Stock Center, Berkeley, CA) wastransformed with either pYES2 or pYES2-TNFrED-Agg using the lithiumacetate method previously described (Gietz,R.D., Schiestl,R.H.,Willems,A.R. & Woods,R.A. Studies on the transformation of intact yeastcells by the LiAc/SS-DNA/PEG procedure. Yeast. 11, 355-360, 1995).Transformed yeast cells were grown overnight in Ura⁻ medium supplementedwith 2% glucose at 30^(o)C with shaking. Expression was induced bygrowing the transformed yeast overnight at 30^(o)C with shaking in Ura⁻medium containing 2% galactose and 1% raffinose. Cells were harvested bycentrifuging at 16,060 x g for 2 min, washing twice with Dulbecco’sphosphate-buffered saline (PBS) (Gibco/BRL) and diluting the cells to 4x 10⁶ cells/ml. 4 x 10⁵ cells (100 ul) were incubated with eitherbiotinylated hTNF-( (50 nM or 10 nM) or goat anti-human sTNF RIantibodies (0.7 ug/ml, R&D Systems) or both for 1 h at room temperaturein a final volume of 140 ul. hTNF-( (Protein Purification group, SeronoReproductive Biology Institute) was biotinylated with the EZ-linkSulfo-NHS-LC-Biotinylation kit (Pierce Corp.). Following the incubation,cells were centrifuged at 16,060 x g for 2 min and re-suspended in 140ul of ice-cold PBS containing 0.1% bovine serum albumin (BSA).FITC-labeled avidin (2.2 ug/ml, Jackson ImmunoResearch) orR-Phycoerythrin-conjugated donkey anti-goat IgG (2.2 ug/ml, JacksonImmunoResearch) or both were added to cells in a total volume of 180 uland incubated at 4^(o)C for 45 min. Cells were centrifuged at 16,060 x gfor 2 min, washed once with ice-cold 1X RDF1 buffer (R&D Systems),re-suspended in ~400 ul of 1X RDF1 buffer and analyzed on BectonDickinson FACSort. The event rate was set at approximately 150 cells/secand a total of 10,000 cells were collected per analysis. The yeastpopulation was gated according to light scatter (size) to avoid analysisof clumped cells. Production and selection of random mutant libraries:

Five unique restriction endonuclease recognition sites were introducedinto the coding region of TNFrED by silent mutagenesis using theGeneEditor in vitro Site-Directed Mutagenesis System (Promega Corp.).This step was completed in order to divide the TNFrED into 6 regions ofbetween 40 to 105 bp. Five of the six regions were separately subjectedto a modification of a random mutagenesis method previously described.Briefly, long oligonucleotides (Midland Certified Reagent Company)spanning a region of TNFrED flanked by unique restriction endonucleaserecognition sites were generated that contain a predetermined amount ofthe three “wrong” phosphoramidites at each position. The amount ofspiked wrong phosphoramidites was adjusted to generate an average ofeither two or three mutations per oligonucleotide. Primers flanking themutated, long oligonucleotide were used in a polymerase-chain reactionto amplify the DNA into cassettes for each region of TNFrED. Randomlymutagenized DNA regions were digested with the appropriate restrictionendonucleases and ligated into the pYES2-TNFrED-Agg construct. Ligationreactions for each mutant library were transformed into XL10-Goldultracompetent cells (Stratagene Corp.) using a ratio of 1 ul ofligation mixture per 70 ul of competent cells following themanufacturer’s protocol. Following the transformation, 20 transformationmixes from each mutant library were pooled and grown overnight at37^(o)C in 500 ml of NZY medium containing 50 ug/ml of ampicillin. Tenrandom clones from each mutant library were sequenced using the ThermoSequenase radiolabeled terminator cycle sequencing kit (AmershamPharmacia Biotech). Approximately 5.0 ug of DNA from each random librarywas transformed into ten aliquots (1 x 10⁹ cells/aliquot) of BJ2168cells using the lithium acetate transformation method. Cells were grownfor 24-30 h at 30^(o)C with shaking. Then approximately 1 x 10⁸ cellswere grown overnight at 30^(o)C with Ura⁻ medium containing 2% galactoseand 1% raffinose for the induction of expression. For each FACSexperiment, 4 x 10⁶ cells were labeled as described above usingbiotinylated hTNF-( at a final concentration of 50 nM for the mutantlibrary containing mutant clone 6 or 10 nM for the other mutantlibraries. Goat anti-human sTNF R1 antibodies were added along withFITC-avidin and R-phycoerythrin-conjugated anti-goat IgG. A total of 1.2x 10⁷ cells was sorted for each library. FACS was completed on a BectonDickinson FACsort with an event rate of <2000 cells/sec. The first roundof sorting was performed in exclusion mode and subsequent sorting wascompleted in single cell mode. Collected cells were seeded intoselection medium with glucose and 1/100 volume was plated forcalculating actual number of cells collected. Selected cells werere-grown at 30^(o)C and then induced in selection medium with galactoseand raffinose for the next round of sorting. Each library was sorted atotal of three to four times. Approximately 0.08%-0.4% of cells werecollected in the first round, and 0.01%-0.2% in the subsequent rounds.The collected cells from the last round of sorting were plated Ura⁻plates to yield single colonies. Recovery and analysis of mutant TNFrEDclones:

Approximately 50 individual yeast clones from each library sort wereanalyzed by flow cytometry. 100 ul of induced cells at 0.15 OD_(600nm)were incubated with either 50 nM or 10nM biotinylated TNF-( in a totalvolume of 140 ul, under same conditions as described above. Binding ofTNF-( was detected with FITC-avidin (2.2ug/ml), and cells were analyzedby flow cytometry on the Becton Dickinson FACsort. Clones having agreater median fluorescence than yeast expressing pYES2hTNFrED-Aggcontrol were chosen for rescue of plasmid. DNA plasmids were recoveredfrom yeast, and transformed into competent E. coli JM109 (Promega Corp.)following the manufacturer’s protocol. Purified plasmid DNA wasre-transformed into BJ2168 yeast cells as described above and individualclones were re-analyzed using methods described above. The TNFrEDregions of positive clones after the re-transformation were sequencedusing the Thermo Sequenase radiolabeled terminator cycle sequencing kit(Amersham Pharmacia Biotech). TNF-(Binding assay

Yeast expressing either the wild-type or mutant TNFrED-agglutinin fusionor yeast containing the control vector pYES2 were resuspended in PBS/BSA (10 mg/ml) at a concentration of 1 x 10⁸ cells/ml. In each well ofa Durapore 96-multi-well plate (Millipore Corp.), 50 ul of the cellsuspension were incubated with 50 ul of PBS/BSA (10 mg/ml) containingvarious concentrations of ¹²⁵I-TNF-( (Amersham Pharmacia Biotech) for 2h at room temperature. Following the incubation, the wells were washedthree times with ice-cold PBS using the MultiScreen filtration system(Millipore Corp.). Non-specific binding was determined with the yeastcontaining the control vector pYES2 and the non-specific binding was<10% of the total counts. The Kd and Bmax was determined using theGraphPad Prism program. Expression of TNFrED mutants.

The pCMVhTNFrED constructs were transiently transfected into HEK293-EBNAcells using a calcium phosphate method. Transfections were completed intriplicate and hTNFrED expression was quantitated from medium harvested48 h post-transfection using an ELISA for hTNF R-1 (R & D Systems).BIAcore Analysis of TNFrED mutants.

Surfaces displaying polyclonal goat anti-human TNFrED were constructedby binding biotinylated antibody (BAF225 from R&D Systems) tostreptavidin-coated Sensor SA chips (P/N BR-1000-32, BIAcore Inc.).Under the conditions of these experiments the streptavidin-biotinylatedanti-TNFrED interaction behaves as if irreversible. Data for thecomparison of TNFrED/TNF-( interaction between mutants and wild-typeTNFrED were collected in HBS (10mM HEPES pH 7.4, 150mM NaCl, 3.4mM EDTA,0.005% P20) as follows.

Conditioned media from transient transfections of HEK293-EBNA cells wereconcentrated with Centricon 10s and the amount of TNFrED protein wasquantitated with the R & D Systems ELISA for hTNF R-1. Buffer-dilutedpurified TNFrED (BS03-99, obtained from IRCS) or buffer-dilutedconditioned medium containing a mutated protein was injected onto aBAF225 surface at 50nM, resulting in the formation of antibody-TNFrEDcomplex. TNF-( at 1 nM (trimer in HBS) was then injected, and kineticsof the binding and dissociation were recorded via the time course of thesurface plasmon resonance response. The BAF-225 surface was regeneratedwith 50% 100 mM sodium citrate pH 2.5/50% 100 mM sodium citrate pH 3.,which stripped off TNFrED and TNF-(. This series of injections wasrepeated for TNF-a at 2, 5, 10, 20, 50, and 100 nM while holding theconcentration of TNFrED injected (wild-type or mutant) at 50 nM. Eachset of binding curves was fit globally using a model for 1:1 interactionthat includes a term for linear baseline drift. Conditioned medium (noTNFrED) and HBS were used as negative controls. The BAF225 surface wasregenerated with 50% 100 mM sodium citrate pH 2.5/50% 100 mM sodiumcitrate pH 3., which stripped off TNFrED and TNF-(. In our hands it hasbeen necessary to have fairly mild regeneration conditions to achieve adata collection rate sufficiently high to make this technology useful.Analysis of crystal structures.

A dataset previously described (Wang,Y., Huq,H.I., de,l.C., X & Lee,B. Anew procedure for constructing peptides into a given Calpha chain. Fold.Des. 3, 1-10 , 1998) was used to analyze the (-( angle distributions ofproline, serine, histidine and isoleucine residues in high-resolutionand non-homologous protein crystal structures. This dataset contains 136x-ray structures with a resolution of 1.8 angstroms or higher, and withthe sequence identity of less than 25% between any pair in the set.There are 803 proline, 1251 serine, 409 histidine and 959 isoleucineresidues in the dataset. The (-( angle space is equally divided into36(36 bins with an interval of 10 degree from –180 degree to 180 degreefor ( or ( angle. The darkness of a bin in the (-( angle distributiondiagrams (Figure 3) is associated with the frequency of binomialdistribution of the amino acid residue in the dataset. The higher thefrequency, the darker the bin. The lightest gray bins represent adimensionless ratio of Z value (fold of probable error over the basalprobability) of 1. The Z value of the darkest (black) bins is 40 orabove, and that of other grey bins is 3, 5, 10 and 30, respectively. Thebasal probability is np, where n is the total number of the amino acidresidue in the dataset and the p is 1/(36(36), and the error variance (is :

Both the above dataset and the Hobohm’s dataset (Hobohm,U. & Sander,C.Enlarged representative set of protein structures. Protein Sci. 3,522-524 , 1994) were used to search for a tri- or a tetra-amino acidsequence corresponding to the same sequence surrounding either themutated residues or the wild-type sequence. The reason to include theHobohm’s dataset for the search is to increase the number of proteinstructures containing the sequence to increase the statisticalrelevance. There are 168 protein structures in the Hobohm dataset, and35 structures are in overlap with the first dataset. The combineddataset contains 269 non-redundant protein structures, which representsthe different type of folds and sequences of the whole PDB database. Thepdb codes of the three crystal structures containing TNFrED moleculesare 1ext, 1ncf and 1tnr . These structures are not in any of the abovedatasets.

The invention has been described in detail with reference to preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements within the spirit and scope of theinvention.

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1. A recombinant mutant of a native polypeptide, wherein the nativepolypeptide has at least one amino acid residue that has angles that arefavored for proline, and wherein said mutant has at least one of saidamino acid residues substituted by proline.
 2. The polypeptide of claim1, wherein the mutant exhibits increased folding efficiency.
 3. Thepolypeptide of claim 2, wherein the substituted amino acid of the nativepolypeptide has a ( angle of –45 to –90 degrees and a ( angle of –60 to0 or 120 to 180 degrees.
 4. The polypeptide of claim 3, wherein thenative polypeptide comprises a cysteine residue capable of forming adisulfide bond, and wherein said substituted amino acid is within 15residues of said cysteine residue.
 5. The polypeptide of claim 4,wherein the substituted amino acid is within 10 residues of saidcysteine residue.
 6. The polypeptide of claim 5, wherein the substitutedamino acid is within 5 residues of said cysteine residue.
 7. A nucleicacid encoding the polypeptide of any one of claims 2-6.
 8. A recombinantmutant of a native polypeptide, wherein the native polypeptide comprisesthe amino acid sequence of SEQ ID NO:1, and wherein amino acid 34 or 87of SEQ ID NO:1 is substituted with proline.
 9. The polypeptide of claim8, wherein the amino acid at position 34 of SEQ ID NO:1 is substitutedwith proline.
 10. The polypeptide of claim 8, wherein the amino acid atposition 87 of SEQ ID NO:1 is substituted with proline.
 11. A nucleicacid encoding the polypeptide of any one of claims 8-10.
 12. Anexpression vector comprising the nucleic acid of claim 7, wherein saidnucleic acid is operatively linked to an expression control sequence.13. An expression vector comprising the nucleic acid of claim 11,wherein said nucleic acid is operatively linked to an expression controlsequence.
 14. A method of producing an isolated mutant of a nativepolypeptide comprising: (a) providing a host cell comprising theexpression vector of claim 12; and (b) expressing the mutantpolypeptide.
 15. A method of producing an isolated mutant of a nativepolypeptide comprising: (a) providing a host cell comprising theexpression vector of claim 13; and (b) expressing the mutantpolypeptide.